Background: We previously investigated the effect of different combined treatments on the tumor microdistribution of Alexa-488-B3, anti-Le-y monoclonal antibody, in a mouse model with the Le-y-positive A431 tumor (200 cubic mm). The treatment regimens we investigated are as follows: 1) a single 150 or 300 micro-g B3 injection (iv), 2) a sequential single injection of 150 micro-g B3 (iv) on day 0 followed by 40 or 70 mg/kg Paclitaxel (ip) on day 1, 3) a sequential single injection of 300 micro-g B3(iv) on day 0 and 70 mg/kg of Paclitaxel (ip) on day 1, and 4) sequential fractional injections involving 150 micro-g B3(iv) on day 0, 40 mg/kg of Paclitaxel (ip) on day 1, 150 micro-g B3 (iv) on day 3 and 30 mg/kg of Paclitaxel (ip) on day 4. The animals were euthanized 2 days after B3 injection and the tumor microdistributions of B3 were analyzed. We then compared two different sets of data obtained from the above experiments; first, the amount (fluorescence intensity) of B3 accumulated to 1 mm from the tumor surface calculated by the area-under-curve (AUC) analysis and second, the tumor microdistribution of B3 expressed as the central to peripheral signal intensity (C/P) ratios of B3. C/P ratios were assessed using following equations; C/P ratio = C / ((P1 + P2 + P3 + P4)/4)), where C was the average of 10 pixel values of the central region of tumor tissue (tumor center, 1.25 - 2.5 mm from the tumor surface; the distance depends on the size of tumor), P1, P2, P3, and P4 were the average of 10 pixel values of peripheral ( 50 micro-m from the tumor surface) tumor tissues taken from two opposite peripheral regions in short axis and long axis, respectively. Results: Compared to the tumor accumulation of the single 150 micro-g B3 dose, 1) a single 300 micro-g B3 dose produced 2.1 fold greater accumulation of B3 in the tumor, 2) 40 and 70 mg/kg of a single Paclitaxel injection following 150 micro-g B3 increased the B3 accumulation by 1.7 and 1.9 fold, respectively. 3) Comparatively, both the sequential single dose and sequential fractional doses increased the B3 accumulation by 3.2 fold. We also obtained the microdistribution data expressed as the central to peripheral signal intensity ratios (C/P ratios) of B3: The 150 micro-g B3 dose produced the B3 concentration in the periphery 3 fold greater than that in the center. Compared to the 150 micro-g B3 dose, the higher B3 dose (300 micro-g) or a sequential single dose (40 or 70 mg/kg) of Paclitaxel did not significantly change the C/P ratios of B3 (due to increase of the B3 concentration in both periphery and center). In contrast, the regimen involving the sequential fractional doses increased the B3 concentration in the tumor center more than that in the periphery, producing the C/P ratio of 1.6 with a more uniform B3 distribution in the tumor than other regimens we investigated. These findings suggest that for 200 cubic mm A431 tumors with a high antigen density (> one million antigens/cell) on the cell surface, a regimen involving the sequential fractional doses could deliver B3 deeper toward the tumor center. -Assessment of spatial correlation between Alexa-B3 distribution and apoptotic cell distribution in tumors after treatment with different combination therapy regimens Objective: In the past year, we investigated if the greater accumulation and deeper penetration of mAb B3 in solid tumor achieved by the sequential fractional doses of B3 and paclitaxel could be explained by the distribution pattern of apoptotic cells (ACs) in tumor. Methods: When the tumor size reached 200 cubic mm, groups of nude mice (n=6/group) were injected as follows: Rx1) a single dose of 300 micro-g Alexa B3 (IV) on Day0 followed by a single dose 70mg/kg paclitaxel (IP) on Day1, and Rx2) Sequential fractional doses of 150 micro-g Alexa B3 on Day0, 40mg/kg paclitaxel on Day1, 150 micro-g Alexa B3 on Day3, and 30mg/kg paclitaxel on Day4, and Rx3) 300 micro-g Alexa B3 (IV) on Day0 as a control. The mice were euthanized 2 or 5 days after Alexa B3 injection. Tumors were harvested with intact skin and flash-frozen. Tumors were fixed with 4% paraformaldehyde overnight at 4 degree C, and were then cryopreserved with 30% sucrose in PBS solution until the tissue was sunk to the bottom of the tube at 4 degree C. Tumors were embedded in OCT (Sakura Finetek) compound for 1 h at -20 degree C prior to frozen sectioning on a microtome-cryostat, and were then stored at -70 degree C. Tumors were sectioned using a Leica CM 1850 cryostat at 8 micro-m thickness to cover the entire tumor tissue and were mounted on the superfrost plus slides. Before staining, slides were warmed at room temperature for 10 min. The tissue slides were rehydrated with 200 micro-l of PBS for 10 min, and were then fixed in 4% paraformaldehyde for 10 min. Apoptosis analysis was performed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling using the Click-iT TUNEL Alexa Fluor imaging assay (Invitrogen). Tissue imaging was performed with 20 objective (pixel size = 0.464 m) using a Scanscope FL (Aperio, Vista, CA) equipped with a motorized scanning stage. Two different channels were obtained: TUNEL assay for apoptotic cells (constant exposure time of 0.4 ms, shown in red) and Alexa B3 antibody (constant exposure time of 1.6 ms, shown in green). Background signal intensity was subtracted to adjust illumination differences for the consistent and repeated scan. Image analysis was performed using in house program MATLAB script (Mathworks, Natick, MA). Individual image channels were exported from Imagescope (provided from Aperio, v11.1.2.760) as TIFF image for the image analysis. Alexa B3 intensity was plotted vs the distance and the B3 accumulation to 1 mm distance from tumor surface was calculated by AUC analysis. B3 intensity across tumor area was obtained. Tumor slices were stained with the TUNNEL reagent to find a correlation between B3 distributions and the apoptotic cell distributions. To find the spatial distributions of B3 and ACs, the individual tissue image for both B3 distributions and AC distributions was segmented using Fuzzy C means clustering. The image correlation was calculated for each segmented image using the in house program. Results: Compared to Rx3 (control), Rx1 increased ACs primarily in tumor periphery (P) with uniform distribution patterns of ACs. The intensity and distribution pattern of ACs from Rx1 showed a positive correlation with those of Alexa-B3 (Pearson correlation coefficient of 0.71 +/- 0.05). Although the single paclitaxel injection improved the total accumulation of B3 by 75% compared to Rx3, it did not significantly change the C/P ratio of B3 (0.250.32 vs 0.340.39 for Rx3), indicating that the apoptosis decreased tumor cell density and improved the accumulation (% ID) of B3 in both P and tumor core (C). In contrast, Rx2 produced very dense clusters of ACs primarily in P with areas of sparse ACs in C. These distribution patterns of ACs resulted in an improved accumulation of B3 by 75% compared to Rx3 and deeper penetration of B3 toward C with its peak intensity shown at 1 mm from the surface and the C/P ratio of 1.54. Conclusion: These findings indicate that a regimen involving the sequential fractional injections (Rx2) is more effective in killing tumor cells in P, thereby reducing the binding-site barrier in P and lowering IFP that resulted in deeper and more uniform distribution of B3 toward the tumor core. This study supports a hypothesis that Rx2 would be a more effective therapeutic regimen than Rx1 for antibody and drug combination therapies of solid tumors.